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In the protein world, structure dictates function. That means the three-dimensional topology of a protein gives important clues to its role in both health and disease. It’s no wonder then that researchers have invested great time and energy performing techniques like X-ray crystallography, which deploys X-ray beams to probe the molecular structure of a protein in its crystal form.

Thanks to IPI’s Rob Meijers and his colleagues at the European Molecular Biology Laboratory, now comes proof of a new structural biology tool. It will enable researchers worldwide to determine the structure of many complex proteins, including those that have previously eluded study.

The story begins as Meijers and his colleagues were working with glycoproteins, proteins with carbohydrates attached. Proteins twist and fold into very specific arrangements. That shaping is often influenced by carbohydrates, or sugars, that latch onto the protein scaffold as it is being formed inside cells. Most proteins that sit on the surface of a cell have such sugars attached to protect them from the rough environment of the extracellular space.

The sugars are critical: they steer protein folding, stabilize structure and route proteins to the correct destination in or outside cells. But some of these sugars, also called N-glycans, can cause insurmountable problems for scientists; the structures can branch elaborately and interfere with protein crystallization in the laboratory.

As a way around the problem, investigators have tried to remove the N-glycans after the protein has folded. Researchers have also attempted to use genetic engineering to prevent the sugars chains from ever being produced or anchored on their glycoproteins. As a consequence, however, the proteins, lacking their sugars, simply do not fold correctly. And everyone goes back to the drawing board.

In 2014, Nico Callewaert’s team at Ghent University in Belgium came up with a clever workaround. The scientists engineered a novel cell line, dubbed GlycoDelete, that churns out proteins with shortened N-glycans. Only three units long, these glycan stumps allow the protein to which they are tethered to fold properly but do not interfere with subsequent crystallization.

“Indirectly, it’s protein design,” Meijers says, “tinkering with a cell line, so you can customize a protein.”

Indeed, Meijers heard about the cell line when he was previously headquartered at EMBL. He and Savvas Savvides at Ghent University pulled together a team to test the cell line’s merit in the manufacturing of three elusive glycoproteins. Meijers studied Down syndrome cell adhesion molecule (DSCAM), overexpressed in Down syndrome patients, and Savvides looked at human colony-stimulating factor 1 receptor (CSF-1R), important in cell signaling, and interleukin 12B (IL-12B), involved in inflammation.

To the researchers’ delight, GlycoDelete cells could express altered DSCAM and CSF-1 molecules. Missing their complex sugars, the glycoproteins better packed into crystals and remained functional. What’s more, for the first time the proteins became amenable to study by X-ray crystallography, yielding new insights into the proteins behavior.

Meanwhile, IL-12B proteins that were expressed by GlycoDelete cells retained their protosugars as they added on the telltale GlycoDelete glycan stumps. This gave evidence that the altered proteins had rerouted to a cell structure called the Golgi. Most importantly, the process allowed the creation of high quality crystals for structural studies of IL-12B.

“Glycoproteins play important roles in development and many diseases,” Meijers says. “Now researchers have one more tool to study them.”

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